It is the overarching hypothesis of this project that long-term therapeutic success against human parainfluenzavirus type 3 (HPIV3) and respiratory syncytial virus (RSV) will require a combination therapy approach with a pair of pathogen-directed inhibitors with distinct mechanistic profile. This notion is driven by the strict safety profile requested by a mostly pediatric patient population and the threat that resistance mutations against individual therapeutics may become fixed rapidly in circulating virus strains. Members of the closely related paramyxo- and pneumovirus families, HPIV3 and RSV are responsible for the majority of severe lower respiratory infection (LRI) and death from viral disease among infants in the United States, and recognized as a potential threat to the immunocompromised and the elderly. Infection by both pathogens initiates in the upper respiratory epithelium, followed by gradual progression to the small airways in patients advancing to severe disease, opening a window for therapeutic intervention. No vaccine protection or effective therapeutic is currently available against either HPIV3 or RSV, and antibody immunoprophylaxis against RSV is restricted to a subset of high-risk patients. This project will address this unmet clinical need by developing applicable, cost-effective therapeutics targeting the viral RNA-dependent RNA polymerase (RdRp) complexes. Building on an established antiviral program, we have recently identified an efficacious nucleoside analog inhibitor with potent activity against both RSV and HPIV3. Serving as reference, this compound will inform the co-development of allosteric RdRp blockers that are rigorously vetted from early stage development for their potential for combination therapy with competitive polymerase inhibitors. In pilot studies, we have engineered a recombinant HPIV3 reporter virus and identified in a high-throughput screening (HTS) campaign using this strain two novel, viable HPIV3 polymerase inhibitor scaffolds with sub-micromolar starting potency. Against RSV RdRp, we have synthetically redesigned an efficacious allosteric measles virus polymerase blocker and identified in the resulting pharmacophore-informed library a potent new point-of-entry with anti-RSV RdRp activity. Recognizing the risk of early stage failure in drug development, we have in parallel identified the protein-protein interface between the RSV polymerase and encapsidated genome as a promising yet underexplored druggable site. To further diversify the portfolio also of allosteric anti-RSV candidates, this site will be interrogated with an innovative biochemical HTS assay and orthogonal counterscreens (aim 1). The existing anti-RSV and anti-HPIV3 leads and newly emerging candidates will be mechanistically characterized using next-generation cell based and in vitro polymerase assays, and subjected to resistance profiling singly and in combination with the reference nucleoside inhibitor (aim 2). Allosteric candidates suitable for RdRp- targeted combination therapy will be synthetically optimized guided by ADME and pharmacokinetic profiles, followed by animal efficacy and toxicity assessment and synergy profiling in cell culture and in vivo (aim 3).